Stable isotope tracer methodology was used to monitor the flux through the de novo pyrimidine pathway in vivo. Recovery of pathway activity after PALA treatment in PALA-sensitive and PALA-resistant tumor lines was determined by quantitating the incorporation of stable-labeled carbon dioxide into uracil nucleotides by GC/MS. Stable-labeled ammonium chloride was found to be an effective precursor for pyrimidine biosynthesis in vivo. Also, in vitro studies demonstrated that ammonium chloride is utilized at the expense of glutamine as a precursor of carbamyl phosphate for the de novo synthesis of pyrimidines in isolated rat hepatocytes. Of twelve new compounds synthesized and tested as inhibitors of uridine kinase, one was found to be a potent inhibitor of uridine utilization by cultured L1210 cells. Riboxamide (NSC-286193) was also found to inhibit uridine utilization by L1210 cells at concentrations that are not growth inhibitory. The regulation of circulating purine concentrations by the isolated perfused rat liver was studied utilizing and artificial oxygen carrier and quantitating of purine uptake and export by HPLC.